Complement activation on immunoglobulin Gcoated hydrophobic surfaces enhances the
release of oxygen radicals from neutrophils
through an actin- dependent mechanism
Jonas Wetterö, Torbjörn Bengtsson and P Tengvall
Linköping University Post Print
N.B.: When citing this work, cite the original article.
This is the authors’ version of the final publication:
Jonas Wetterö, Torbjörn Bengtsson and P Tengvall, Complement activation on
immunoglobulin G-coated hydrophobic surfaces enhances the release of oxygen radicals from
neutrophils through an actin- dependent mechanism, 2000, Journal of Biomedical Materials
Research, (51), 4, 742-751.
http://dx.doi.org/10.1002/1097-4636(20000915)51:4<742::AID-JBM24>3.0.CO;2-D
Copyright: Wiley
Postprint available at: Linköping University Electronic Press
http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-25790
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
Complement activation on immunoglobulin G-coated hydrophobic
surfaces enhances the release of oxygen radicals from neutrophils
through an actin-dependent mechanism3
Jonas Wetteröa, b, Torbjörn Bengtssonb and Pentti Tengvalla
a
Laboratory of Applied Physics, Department of Physics and Measurement Technology,
Linköping University, SE-581 83 Linköping, Sweden
b
Division of Medical Microbiology, Department of Health and Environment, Faculty of
Health Sciences, Linköping University, SE-581 85 Linköping, Sweden
Correspondence to: Jonas Wetterö (e-mail: [email protected])
Key Words: neutrophil respiratory burst, IgG, complement, wettability, actin cytoskeleton
3
No benefit of any kind will be received either directly or indirectly by the authors
1
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
Abstract: Neutrophil granulocytes are among the first cells to encounter a plasma protein-coated
implant and may through frustrated phagocytosis release toxic oxidative species. We utilized two
model surfaces, hydrophobic and hydrophilic glass, to investigate the effects of plasma
immunoglobulin G (IgG) - complement interactions for neutrophil adhesion and respiratory burst.
The respiratory burst was measured with luminol-amplified chemiluminescence (CL) and cell
adhesion was determined by labeling
neutrophils with 2´, 7´-bis-(carboxy-ethyl)-5(6)-
carboxyfluorescein (BCECF). We demonstrate that the IgG-triggered neutrophil adhesion and
oxygen radical production is augmented in the presence of normal human serum (NHS), in
particular on hydrophobic surfaces,indicating that complement factors enhance the neutrophil
activation. We propose that the complement factors C3, C5a and C1q are especially important for
this amplification, but factor B is probably not. Disturbance of the actin filament dynamics with
cytochalasin B or jasplakinolide blocked the neutrophil radical generation on all surfaces.
However, these drugs did not affect the number of adherent neutrophils. We suggest that there is a
synergistic interaction between adsorbed IgG, and the complement system , which amplifies the
neutrophil acute inflammatory responses through a dynamic actin cytoskeleton on synthetic
surfaces.
2
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
INTRODUCTION
An artificial surface that encounters blood becomes coated with plasma proteins e.g. fibrinogen,
albumin and immunoglobulin G (IgG) within seconds.1 The role of plasma proteins in the host
recognition of implants is poorly characterized, but it is evident that the surface chemistry and
wettability strongly influence the composition of the adsorbed layer. Vroman exchange-like
phenomena are reported at hydrophilic surfaces2 and fibrin/fibrinogen appears to be of great
importance in mediating the acute inflammatory response at hydrophobic surfaces.3 Albumin is
suggested to blunt the inflammatory response4 and adsorbed IgG is known to activate and recruit
blood cells in vitro4,5 and in vivo.6
The activation of complement during blood-biomaterial interactions is a clinically observed
phenomenon,7-11 although the mechanisms are not fully understood. Probably both the classical and
the alternative complement pathways are involved. In brief, the classical activation is initiated by
immunoglobulins and complement factor 1q (C1q) leading to deposition of C3b and activation on the
target surface. The alternative complement activation is triggered by anon-specific binding of C3b.
Both pathways serve to form C3-convertases that cleave C3 to form C3a and C3b.12 The binding of
complement to artificial surfaces may also be affected by other plasma proteins.13
Neutrophils can be found on artificial surfaces within minutes of blood contact.14-16 The cell migration
is initiated by a local release of chemotactic stimuli, for instance complement components in a
damaged tissue. The neutrophil phagocytosis of a prey involves fixation of the target to the neutrophil
surface. Most often this is accomplished through the interaction between opsonizing complement
residues on the target surface and complement receptors on the neutrophil plasma membrane.
Neutrophils carry complement receptors (CR1 and CR3) that bind C1q, C3b and iC3b. However, the
3
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
fixation is not complete unless a second activation occurs from either bacterial carbohydrates or
opsonising IgG (ligand for neutrophil Fcγ-receptors II and III).17 Apart from enhancing the neutrophil
adhesiveness and the phagocytic behavior on biomaterial surfaces,18 adsorbed IgG induces
the
neutrophil generation of oxygen free radicals.5 Furthermore, immobilized IgG has been suggested to
activate C3 in serum and, accordingly, increase the C3b and iC3b depositions onto both hydrophobic
and hydrophilic surfaces.19
The microbicidal activity of neutrophils can be divided into mechanisms dependent or independent
on oxygen.20,
21
The oxygen-dependent mechanisms consume oxygen as an electron acceptor in a
reaction initiated by the activation of a multicomponent electron transfer system, the NADPHoxidase. This way relatively atoxic superoxide anions are produced which can be dismuted by
superoxide dismutase (SOD) to antimicrobical hydrogen peroxide. In parallel with the generation of
toxic oxidative products, protective antioxidant systems are activated. For instance, catalase produces
water and oxygen from hydrogen peroxide. The neutrophil respiratory burst is often studied with
luminol-amplified chemiluminescence (CL).22
The neutrophil spreading and migration over a surface requires rearrangements of the actin
cytoskeleton, including reversible transformations between globular and filamentous (F) actin. Actin
filaments organized in adhesion sites (focal contacts) and pseudopodia are considered to play a
critical role in neutrophil behavior by regulating the dynamic interactions between β 2-integrins and
extracellular matrix proteins. Several findings also indicate that secretion of hydrolytic enzymes and
production of oxygen metabolites are directly regulated by the actin filament system.23-25 In support,
an association between components of the NADPH-oxidase, β 2-integrins (e.g. CR3) and actin
cytoskeletal structures has been reported.26, 27
4
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
In the present study we used a well defined clinically relevant biomaterial wettability model-system5,
28-30
in order to elucidate the effects of IgG on neutrophil respiratory burst and adhesion and its
correlation to complement activation and actin rearrangements.
MATERIALS AND METHODS
Materials
All reagents were of analytical grade and purchased from Sigma Chemical Co. (St. Louis, MO, USA)
if nothing else is denoted. Cell separation products dextran and Ficoll-Paque were from Pharmacia
Fine Chemicals (Uppsala, Sweden). Sodium-metrizoate was delivered from Nycomed Pharma (Oslo,
Norway). CL reagents horseradish peroxidase, SOD and catalase were from Boehringer Mannheim
GmbH (Mannheim, Germany). Normal human IgG (Gammaglobulin 165 mg/ml) was from KabiPharmacia (Uppsala, Sweden). In the CL assay four ml disposable polypropylene tubes (Sarstedt
GmbH, Nümbrecht, Germany) and 40 x 8 mm glass test tubes with a high SiO2 content (Assistent
KHG, Sondheim-Rhön, Germany) were used. Complement-depleted sera and purified complement
components were from Quidel Corp. (San Diego, CA, USA). Giemsa staining solution was from
Merck (Darmstadt, Germany). Bodipy-phallacidin and jasplakinolide were purchased from Molecular
Probes Inc. (Eugene, OR, USA). The antibodies M 0741 (Monoclonal Mouse Anti-Human C3bi
Receptor, CD11b, clone 2LPM19c), X 0931 (Mouse IgG1 Negative Control), A 0062 (Polyclonal
Rabbit Anti-Human C3c Complement) and A 424 (Polyclonal Rabbit Anti-Human IgG, Specific for γChains) were from Dakopatts (Glostrup, Denmark). Azide free monoclonal F(ab´)2:s RDI-023-301
(mouse anti-human CD 32, clone IV.3), RDI-028-201X (mouse anti-human CD 16, clone 3G8) and
mouse IgG2 isotype control antibody RDI-MSIGG2A-X (clone G155-178) were from Research
Diagnostics, Inc. (Flanders, NJ, USA). Monoclonal mouse anti human CD88 (MCA1283XZ, clone
S5/1)
came
from
Serotec
(Oxford,
England).
Purified
(>80%)
P1-peptide
(H-
KYGWTVFQKKRLDGSV-OH) was from Research Genetics (Huntsville, AL, USA). 2´, 7´-bis-
5
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
(carboxy-ethyl)-5(6)-carboxyfluorescein
pentaacetoxymethyl
ester
(BCECF-AM)
was
from
Calbiochem (La Jolla, CA, USA).
Neutrophils
Peripheral blood was drawn daily from apparently healthy volunteers into heparinized tubes. The
erythrocytes were removed by dextran-sodium metrizoate (2:1) sedimentation at 1 x g, followed by 35
seconds of hypotonic lysis in ice-cold distilled water. The neutrophils were then separated from
monocytes, lymphocytes and platelets by Ficoll-Paque centrifugation (400 x g, 4°C) and washed twice
(200 x g, 4°C) in Krebs-Ringer phosphate buffer, pH 7.3, supplemented with 1.5 mM Mg2+ and 5mM
glucose.31 The washed neutrophils were then resuspended in the previous buffer with an addition of 1
mM Ca2+ (KRG) and counted in a Coulter Counter (Coulter Electronics, Ltd., England). The isolated
neutrophils were kept on ice until the experiments were performed within a few hours after the
preparation. Morphological studies showed no signs of cell activation due to the preparation
procedure, a high cell viability and very low contamination of other blood cells. Control experiments
were performed with neutrophils isolated via alternative preparation techniques from citrated whole
blood and buffy coats, as well as by using diluted heparinized whole blood.
Sera
Normal human serum (NHS) was prepared by clotting and centrifugation of fresh whole blood from
three AB-positive donors at room temperature. In some experiments, the NHS was decomplemented
by heat-inactivation for 30 minutes at 56°C. Both types of sera were filtered through sterile 0.22 µm
Millipore Millex-GV filters in order to remove cellular debris. Commercially decomplemented NHS
depleted on or deficient32 in complement components C1q, C3 and B, respectively, were also used.
The functional activity of these was restored by addition of the purified components. All types of sera
were frozen below -70°C immediately after preparation. Most often, a final serum concentration of
5% (v/v) was used. Control experiments with heparinized or citrated plasma were also performed.
6
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
Importantly, frozen NHS elicited the same CL response as the freshly prepared, and the sterile
filtration of the NHS did not interfere with the study.
Hydrophobic and hydrophilic surfaces
Glass test tubes were cleaned for five minutes at 80°C in a solution consisting of five parts of distilled
water, one part of hydrogen peroxide (30% v/v) and one part of NH4OH (25% v/v). After repeated
rinsing in distilled water, the tubes were washed for five minutes at 80°C in another solution
composed of six parts of distilled water, one part of hydrogen peroxide (30% v/v) and one part of
hydrochloric acid (37% v/v). This procedure covers the surface with a thin, hydrated, layer of SiO2
and generates a water/surface advancing contact angle θw<10° (measured with a Ramé-Hart NRL
Model 100 goniometer). Some of the hydrophilic tubes were saved and stored in an acid solution. The
other tubes were washed in distilled water, technically pure ethanol and trichlorethylene, and
methylated through five minutes of incubation at room temperature in trichlorethylene with 1% (v/v)
dichlorodimethylsilane added. Traces of silane was removed through excessive rinsing in ethanol,
trichlorethylene and, finally, ethanol. The hydrophobic preparations were stored in ethanol for not
more than a week and the hydrophobicity was continuously checked (θw>90°).
Coating with IgG
A stock solution of 10 mg IgG per ml was prepared in phosphate buffered saline, pH 7.3
(PBS). The normal human IgG used was derived from the plasma pool of approximately
10,000 European blood donors and should have an immunoglobulin A contamination of no
more than 0.05%. The purity of this product was checked via reduced SDS-PAGE.
Hydrophobic and hydrophilic tubes were washed twice in PBS, then 1 mg/ml of IgG in PBS
was incubated in the test tubes for 30 minutes at room temperature. Before the final sample
solutions were added, the tubes were rinsed twice in PBS. By this procedure, a monolayer of
7
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
IgG was adsorbed to the glass surfaces. The antigenicity was controlled in experiments using
similar silicon surfaces, antibody A 424 and single wavelength ellipsometry in air (on a
Rudolf Research AutoEl, NJ, USA, with a HeNe λ=6328 Å laser) in order to determine the
thickness of the adsorbed monolayer (approximately 20 Å). In some experiments, surfaces
were coated with human serum albumin as a negative reference to IgG (approximately 12 Å).
Neutrophil radical production
Glass test tubes (IgG- or non-coated) with total sample volumes of 0.5 ml in KRG were placed inside
four ml polypropylene tubes.5 NHS was added to some samples, and all solutions were mixed and
allowed to equilibrate at 37°C for a couple of minutes. After this, CL reagents and neutrophils were
added. Luminol (5-amino-2,3-dihydro-1,4-phtalazinedione, 50 µM) and extra peroxidase (horseradish
peroxidase, 4 U/ml) were used for the determination of the total oxidase activity. The intracellular
response was quantified using superoxide dismutase (200 U/ml) and catalase (2,000 U/ml) instead of
extra peroxidase. The CL was registered for half an hour at 37°C in a just calibrated six channel
Biolumat LB 9505 C from Berthold Co. (Wildbaden, Germany). The actin polymerization inhibitor
cytochalasin B (12,5 µg/ml), or stimulator jasplakinolide (0.1-10 µM), was added to, or pre-incubated
with, the sample solutions in some experiments. In other experiments, the neutrophils were incubated
with the CR3-blocking33,
34
antibody M 0741, or with the P1-peptide (100 µM), using the X 0931
antibody as a negative control. To elucidate a possible priming role of the anaphylatoxin C5a, the
neutrophil C5a-receptor (CD88) was blocked with the azide free MCA 1283XZ antibody and
compared with the response from cells treated with the control antibody RDI-MSIGG2A-X.
Adhesion, morphology and F-actin distribution
The number of adhered neutrophils was determined after 30 minutes of incubation inside the MultiBiolumat using several methods. First, the number of cells remaining in the cell was counted using a
Coulter Counter ZM Channelyser 256 (Coulter-Electronics, Luton, UK). Secondly , neutrophils were
fixed on slides and stained, counted and studied morphologically. The cells were fixed for 15 minutes
in methanol for nuclear staining and incubated for 15 minutes in a Giemsa/methanol (4 + 25 parts,
8
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
v/v) solution on ice, after which they were viewed by light microscopy. F-actin was stained and
studied in a fluorescence microscope after 30 minutes of fixation in ice-cold paraformaldehyde (4%)
and
30
minutes
incubation
in
a
mixture
of
bodipy-phallacidin
(0.6
µg/ml)
and
lysophosphatidylcholine (100 µg/ml) in PBS protected from light.35 Bodipy-Phallacidin stained
samples were also documented with a Sarastro 1000 confocal laser scanning microscope from
Molecular Dynamics (Sunnyvale, CA, USA). This microscope had a Nikon x 100 (1.4 N.A.) objective
and the samples were excited at 488 nm by the Argon laser. The laser intensity and the
photomultiplier voltage were kept constant throughout the photo sessions. As a third approach, the
neutrophils were labeled with 4 µM BCECF-AM (40 minutes in 37 °C) and washed twice in KRG
(200 x g, 4°C). The labeled cells were allowed to adhere to the surfaces, non-adherent cells were
rinsed off and the remaining neutrophils were lysed with 0.5% (v/v) Triton X-100.36, 37 The number of
adhered neutrophils were then calculated by excitation of the samples at λ=439 nm and the
fluorescence was measured at λ=505 nm in a Perkin-Elmer LS-3B spectrofluorometer (Perkin-Elmer
Ltd., Beaconsfield, Buckinghamshire, England). The fluorescence emanating from the adhered
neutrophils was divided with the total fluorescence from free and surface-associated cells. The
autofluorescence from non-labeled cells was routinely subtracted.
Statistics
Data from the experiments are expressed as the mean values ± standard errors of the means (SEM).
Students two-tailed T-test for paired observations was used for the comparison between activated
cells and control cells. Differences with p<0.05 were regarded as significant.
RESULTS
The effect of wettability on IgG immobilization and the neutrophil respiratory burst
The exposure of neutrophils in KRG to pure hydrophilic glass rendered a more pronounced oxygen
radical production than cells subjected to a hydrophobic surface. The average CL of neutrophils from
9
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
19 donors run in duplicate was 1,78 ± 0,12 x 108 counts per minute (CPM) on hydrophilic, and 1,36 ±
0,11 x 108 CPM on hydrophobic surfaces (p<0.001). Pre-adsorbed IgG appeared to slightly enhance
the respiratory burst on both types of surfaces, although the CL responses did not differ significantly
from those of control cells (Figure 1). The CL responses, that included both extra- and intracellular
oxygen radical production, were monophasic with a peak after approximately five minutes and
declined within an hour. However, pre-coating of either surface with IgG augmented the radical
production in whole blood (Table 1). Ellipsometric analysis using the antibody A 424 indicated that
the IgG-monolayers were stable for days on hydrophobic silicon, whereas the antigenicity declined
within hours after preparation on hydrophilic silicon. Furthermore, an increase of the IgGconcentration during the coating procedure did not further amplify the CLresponse.
Complement activation on hydrophobic and hydrophilic glass
The CL response in neutrophils interacting with IgG on hydrophobic or hydrophilic surfaces was
augmented by the addition of NHS (Figure 1). The maximal CL-response was obtained with 5 to
10% (v/v) NHS and the response declined within an hour. Studies with superoxide-dismutase and
catalase indicated that mainly the extracellular release of oxygen metabolites was enhanced. The
NHS-amplified CL response on the IgG-coated hydrophobic surfaces included a marked second
phase after approximately 20 minutes. The second phase disappeared when NHS was heat-inactivated,
p<0.001 (Figure 2), or when a C3-depleted serum was used. Furthermore, when the C3-depleted
serum was reconstituted with C3, a response similar to that observed with NHS was restored (Figure
3). The “IgG/NHS-effect” could be quenched by rinsing the NHS preparations in KRG prior to the
addition of cells. Ellipsometric investigation with antibody A 0062 on IgG-coated hydrophobic silicon
surfaces revealed the deposition of C3 almost instantly after exposure to NHS, although no C3 was
found when heat-inactivated NHS was used instead (not shown). Blocking of the neutrophil receptors
CR3 (antibody M0741), FcγRII (fragment RDI-023-301) and FcγRIII (fragment RDI-028-201X) did
not alter the IgG-triggered CL response in the presence of NHS. However, blocking of CR3 with the
P1-peptide, reduced the CL by approximately 50-75%. The factor B-depleted serum behaved similar
10
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
to NHS. Somewhat surprising, C1q-depleted serum was found to enhance the neutrophil respiratory
burst biphasically when exposed to IgG-coated hydrophobic glass. The oxygen radical production was
further amplified with the reconstitution of C1q to the depleted serum (Figure 4A). Blocking of CD88
reduced the overall “IgG/NHS-effect” by 22.7% (p<0.05, Figure 4B) and the IgG-triggered neutrophil
response to heat-inactivated serum was lowered by 12.9% (not significant). Incubation with the
negative control antibody showed no detectable effect on the CL-response.
In order to elucidate the possible participation of the most abundant plasma proteins, we added normal
or heat-inactivated albumin, fibrinogen or IgG, respectively, into non- and IgG-coated hydrophobic
glass tubes. Stimulatory effects similar to those of NHS were not obtained. A phenomenon similar to
that elicited by NHS was observed when citrated platelet poor or platelet free plasma were exposed to
IgG-coated hydrophobic glass.
The enhancement of the neutrophil oxygen radical production by the combination of IgG and NHS
was much less pronounced on the hydrophilic compared to on the hydrophobic glass (Figure 2). The
amplification was monophasic and disappeared when using heat-inactivated NHS instead (Figure 3).
Interestingly, heat-inactivated NHS markedly increased the neutrophil generation of oxidative species
on the hydrophobic surfaces , but non-heated NHS did not trigger the neutrophil respiratory burst.
Neutrophil adhesion
The NHS-mediated amplification of the IgG-induced CL response originated mainly from
adheredneutrophils, since the major part of the response remained when the non-adherent
cells were rinsed off the surfaces. In order to elucidate if the oxygen radical production was
correlated to the number of adherent neutrophils, an adhesion study was performed. Noncoated hydrophobic and hydrophilic surfaces attracted similar numbers of cells. IgG-coated
hydrophobic surfaces attracted approximately 30% more neutrophils with NHS present than
the NHS-free surfaces (Figure 5). Heat-inactivation of NHS did not affect the adhesion to
11
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
hydrophobic glass. It was found that IgG-coating on hydrophilic glass, with or without added
NHS, had no effect on the number of adhered cells. Consequently, no direct correlation was
found between the number of adherent neutrophils and the generation of oxygen metabolites.
Morphological studies of Giemsa or bodipy-phallacidin stained neutrophils revealed similar
results regarding the number of adhered cells, however showed that cells on both non- and
IgG-coated (with or without NHS) surfaces displayed a more spread out morphology than
cells treated with NHS only (Figure 6). Role of F-actin
The spreading and movement of neutrophils on a surface is highly dependent on a dynamic actin
cytoskeleton, including reversible polymerization of actin filaments. We found that neutrophils spread
out through an extensive actin polymerization in protruding lamellipodia and pseudopodia on IgGcoated hydrophobic surfaces, especially when NHS was present. Although a hydrophobic surface
exposed to NHS supported the adhesion of neutrophils, the extent of spreading and actin
reorganization was much more pronounced on the clean or IgG-coated surfaces (with or without
NHS). Together with the CL data, these observations suggest a correlation between the cell spreading
and oxidase activation. In order to explore this further, neutrophils were treated with cytochalasin B
or jasplakinolide, an inhibitor and a stimulator, respectively, of actin polymerization. Although
neutrophil adhesion was not affected by cytochalasin B and jasplakinolide, these drugs markedly
inhibited theoxidase activation, actin polymerization and cell spreading, . The oxygen radical
production on the different surfaces was totally blocked by the treatment of the neutrophils with
cytochalasin B or jasplakinolide. Exposure of adherent neutrophils to cytochalasin B or jasplakinolide
instantly turned off the oxygen radical production that reached the base levels within seconds.
Cytochalasin B- and jasplakinolide-treated neutrophils that encountered the hydrophobic "IgG/NHSsurface" exhibited a reduced, but still obvious cell spreading. However, the organization of the actin
cytoskeleton was clearly disturbed. Instead of a peripheral localization of F-actin, concentrated in
pseudopodia, actin filaments were found mainly in the central parts of cytochalasin B-treated
neutrophils (Figure 6). In jasplakinolide-treated neutrophils, the F-actin was localized to surface
“blebs”.
12
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
DISCUSSION
The biomaterial surface wettabilityis an important factor for the activation of adhering neutrophils,
probably by eliciting variation in the adsorption of proteins from complex solutions such as blood
plasma. The main finding in the present work is the complement and F-actin dependent frustrated
phagocytosis accompanied by a massive radical leakage on IgG-coated hydrophobic glass upon
incubationin the presence of NHS. This phenomenon did not take place on any of the hydrophilic
preparations.
We found that surface-bound IgG triggered a release of oxygen metabolites from neutrophil
granulocytes. This is in correlation with previous in vivo6 and in vitro4, 5 studies. Ellipsometric data
from hydrophobic silicon surfaces showed that the IgG surface concentration was retained at a
consistent level for days , but declined within hours on hydrophilic silicon. This was also consistent
with our CL-data in whole blood, where the stable results on hydrophobic glass suggested a surfacemediated activation, and the scattering data on hydrophilic glass indicated a bulk-related
phenomenon. From the above results we conclude that adsorbed IgG remains native enough to
opsonize the model surfaces. A large IgG-binding implant area is thus likely to enhance the neutrophil
respiratory burst via a Fc-receptor-mediated pathway and possibly induce frustrated phagocytosis with
the release of reactive oxygen species close to the implant and surrounding tissues. It remains obscure,
however, whether this frustrated phagocytosis is a positive or negative occurence for the tissue
integration of a biomaterial in vivo.
The addition of NHS markedly amplified the IgG-triggered neutrophil respiratory burst in a biphasic
manner. Upon the formation of an antigen-antibody complex, the classical complement activation
pathway may be triggered with the subsequent formation of several components known to enhance
neutrophil adhesion, chemotaxis and respiratory burst. Recently, immobilized IgG was suggested to
13
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
initiate classical complement activation via binding of C1q from serum on methylated silicon. 38, 39
Both C1q and IgG can be detected by antibodies on IgG-coated hydrophobic silicon surfaces during
serum incubations for up to five minutes. C3 is detected by antibodies on these surfaces for at least an
hour. It has been proposed that C1q triggers the neutrophil superoxide production by a unique CD18dependent mechanism.40 The role of C1q and the classical complement activation pathway were
supported by the observation that the IgG-triggered oxidative response was amplified upon
reconstitution of C1q to the C1q-depleted serum. However, the activation of the classical complement
activation pathway can not be the only explanation for the observed CL response, since the addition
of C1q to the depleted serum was not a prerequisite for the amplification to occur. Also, regardless of
IgG coating, a
substantial radical production was observed in hydrophobic tubes when heat-
inactivated NHS was used, despite the fact that C1q is thermolabile.32 Consequently, the role of C1q
and the classical activation pathway needs further investigation. Since factor B-depleted serum
evoked a response similar to that of NHS after exposure to IgG-coated hydrophobic surfaces, we
suggest that the alternative complement activation pathway plays a minor role for the rapid
amplification of the IgG-triggered neutrophil respiratory burst.
Results from experiments with C3-depleted serum and data from Tengvall et al. (1996 and 1997)38, 39
support the hypothesis that C3 deposits on adsorbed IgG, thereby promoting further complement
activation and/or amplification of the neutrophil adhesion and the respiratory burst. In another study,
the presence of C3 in NHS was shown to be important for the neutrophil adhesion to polymer
surfaces.41 It was also demonstrated that solid surfaces, like mercaptoglycerol-modified gold, may
cause a spontaneous complement activation through the increased production of iC3b and the
membrane attack complex (MAC), as well as elevated deposition of C3b during incubation in NHS.38,
39, 42
Fragments of C3b and iC3b that were bound to materials, e.g. latex, have in other studies been
suggested to increase neutrophil oxygen consumption.43 Karlsson et al. (1996) found that C3 adhered
to hydrophobic surfaces, and this was accompanied by an increased respiratory burst and elevated
expression of the surface integrin CD11b/CD18 (CR3) in neutrophils.30 Also, a priming phenomenon
14
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
due to immobilization of C3 was observed, possibly due to the release of the chemotactic complement
factor C5a from the hydrophobic surface. Yet another team, that worked with polystyrene-associated
monomeric IgG, found attenuation of the neutrophil HOCl-generation and collagenase activation upon
exposure to NHS. This was accompanied by an extracellular release of azurophilic MPO and
cathepsin G.44 These effects were not seen when complement-inactivated sera were used instead of
NHS and the authors suggested that C3-fixation by IgG was the primary cause for these events.
CR3 has been suggested to enhance FcγR-mediated signal transduction and adhesion of neutrophils.45
The blocking of CR3 in this study with the P1-peptide reduced the CL response, suggesting that iC3b
was directly involved in the neutrophil radical generation. We know from the literature that iC3b is
incapable of creating a respiratory burst by itself, but in combination with stimulation of neutrophil
FcγRIII, a synergistic enhancement of the neutrophil oxygen radical production occurs.46 In order to
show such a synergistic receptor effect in our system it is necessary to block both types of neutrophil
receptors. Further testing is also required to elucidate the role of CR1 stimulation through surfacebound C3b since C3b is a bivalent molecule47 and IgG has specific attachment sites for C3b.48 The
comparably low responses on the hydrophilic surfaces probably was due to the low protein retaining
capacity of the hydrophilic surfaces; i.e. IgG was probably removed by the serum from the surface out
to the liquid phase.
The presence of NHS alone , without surface-bound IgG, down-regulated or had no effect on the
neutrophil respiratory burst. In contrast, upon exposure of clean hydrophobic surfaces to heatinactivated serum the radical generation was more than doubled, but almost totally quenched in the
hydrophilic tubes (Figure 1). Therefore, it should be stressed that even though heat-inactivation of
NHS inhibits the complement cascade, several soluble complement components are not thermolabile.
For instance, components C3a, C4a and C5a have high degree of α-helical conformation and each
contains three disulfide bridges. Consequently, they resists the heat treatment (56°C for 30 minutes)
used in this study.49 However, in this study we found that liberation of C5a from the IgG-coated
15
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
surfaces, subsequently followed by neutrophil priming, could not be the only explanation for the very
potent radical responses observed in the presence of either normal or heat-inactivated serum, since the
effect of CD88-blocking was moderate. Yet another explanation is the heating process of the NHS
that, theoretically, could result in the formation of various activating protein residues and aggregates,
as well as providing the physiological temperature range for a limited time, where the opsonising
component iC3b is known to spontaneously form in isolated serum due to the autodegradation of C3.
Furthermore, a complex medium such as NHS could of course activate the neutrophils through
surface activation of other plasma protein systems than complement.
The neutrophil spreading and migration over a surface is accompanied by a cyclic regulation of
integrin adhesivity and actin polymerization. In this study, we also demonstrated a correlation
between the extent of cell spreading and the amount of oxygen radicals that was produced. The
disturbance of actin dynamics by pre-treatment of neutrophils with cytochalasin B or jasplakinolide
showed no influence on the number of adherent cells on IgG/NHS-surfaces, but dramatically inhibited
cell spreading, pseudopodia formation and the production of oxygen radicals. This probably reflects
inhibition of the actin-dependent clustering of integrins, formation of adhesion sites and associated
assembly of oxidase components. The introduction of cytochalasin B or jasplakinolide to adhered and
oxygen radical producing neutrophils induced instantaneous reorganization of the actin cytoskeleton
and turn-off of the respiratory burst. However, the adhesion and spreading on IgG/NHS-surfaces were
more or less unaffected. Liu et al. (1997) reported a similar effect on the respiratory burst when
cytochalasin B was added to adhered neutrophils, although they interpreted the oxygen radical turnoff as an indication of dependency of cell adhesion for radical production.5 We suggest that a
continuos interaction between integrins and the actin cytoskeleton plays a central role for the
generation of intracellular signals in adhering cells and is a prerequisite for a maintained respiratory
burst. In correlation, Zhou and Brown (1994) have shown that cytochalasin B counteracts the
synergism between Fc-receptors and complement receptors for the activation of the NADPH-
16
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
oxidase.46 They suggest that ligation of CR3 stimulates the association of FcγRII with the actin
cytoskeleton in the adherent neutrophils, leading to an enhanced respiratory burst.
We conclude that adsorbed IgG and the associated complement activation involving C1q, C3 and C5a
in NHS, amplifies the neutrophil inflammatory responses, particularly on hydrophobic surfaces. The
increased respiratory burst is accompanied with neutrophil spreading and extracellular release of
oxygen radicals and is regulated by a dynamic actin cytoskeleton.
ACKNOWLEDGEMENTS
Ms Agneta Askendal, Dr. Stefan Zalavary, Mrs Kristina Orselius and Mr Mats Wolving are
acknowledged for support. This study was financed by the Swedish Biomaterials Consortium funded
by the Swedish National Foundation for Strategic Research, SSF, and by grant number 19X-12668
from the Swedish Medical Research Council.
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LEGENDS TO TABLE AND FIGURES
21
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
Table 1: Effects of immobilized IgG on the radical production in diluted whole blood
Luminol-amplified CL was measured in hydrophobic and hydrophilic glass tubes, with or
without IgG-precoating, at 37 °C for 30 minutes in a Multi-Biolumat. 4U/ml horseradish
peroxidase was added. The total amount of counts per minute was observed and expressed as
per cent of the response at non-coated surfaces ± standard error of the mean (SEM). The
experiments were performed in duplicate with three or four donors.
Figure 1. Effect of surface-bound immunoglobulin G (IgG) on the neutrophil
respiratory burst in the absence or presence of normal human serum (NHS). Luminolamplified chemiluminescence (CL) was measured in (A) hydrophobic or (B) hydrophilic
glass test tubes with or without 5% (v/v) normal or heat-inactivated serum and 1 x 106
neutrophils per ml KRG at 37 °C for 30 minutes. Extra peroxidase was added (4U
horseradish peroxidase per ml). The results, based on the total amount of counts per minute,
are expressed as percent of control (non-coated hydrophobic or hydrophilic glass) and
represents the mean values from separate experiments with 12 (A) or 9 (B) donors run at
least in duplicate. Bars indicates the standard errors of the means (SEM). For absolute values
of the controls, see text. On hydrophobic glass, “IgG and NHS” (p<0.001), “IgG and Heatinactivated serum” (p<0.001) and “Heat-inactivated NHS” (p<0.01), and on hydrophilic
surfaces “NHS” (p<0.01), “Heat-inactivated NHS” (p<0.001) and “IgG and Heat-inactivated
serum” (p<0.001), were found to differ significantly from control cells.
Figure 2. Chemiluminescence (CL) profiles that show representative kinetics for the actual
time traces of CL from 1 x 106 neutrophils/ml in KRG during 30 minutes of incubation in
37°C with HRP (4U/ml), luminol (10-5M) and NHS (5%) or heat-inactivated NHS (5%)
added to IgG-coated hydrophobic or hydrophilic glass tubes. An instantaneous radical turnoff occurs when jasplakinolide (5 µM) or cytochalasin B (12.5 µM) are added (illustrated
22
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
with the CL-profile after addition of jasplakinolide at time = 15 minutes). The CL was
registered as counts per minute (CPM) and the neutrophils were isolated from the same donor
(exept for the cells treated with jasplakinolide).
Figure 3. Effects of surface-bound Immunoglobulin G (IgG) on the neutrophil
respiratory burst in the absence or presence of C3-depleted serum. Luminol-amplified
chemiluminescence (CL) was measured in hydrophobic glass test tubes with 5% (v/v) C3depleted serum and 1 x 106 neutrophils per ml at 37 °C for 30 minutes. Extra peroxidase was
added (4U horseradish peroxidase per ml). The results, based on the total amount of counts
per minute, are expressed as percent of the response at non-coated hydrophobic surfaces and
represents the mean value of separate experiments with 4 donors run in duplicate. Bars
indicate the standard errors of the means (SEM). “IgG/C3-depleted serum” differs
significantly from “IgG/C3-depleted serum + C3” (p<0.05).
Figure 4. (A) Reconstitution with C1q (180 µg/l) in the C1q-depleted serum further potentiates the
total IgG-triggered neutrophil respiratory burst on hydrophobic glass (p<0.05). (B) Blocking of the
CD88 through 30 minutes of pre-incubation at 4°C with 10 µg/ml of the azide free MCA 1283XZ
antibody significantly (p<0.05) lowers the hydrophobic “IgG/NHS-effect”. Similar incubation with
the
control
antibody
RDI-MSIGG2A-X
showed
no
effect
on
the
luminol-amplified
chemiluminescence (CL) response. Counts per minute (CPM) data represents the total CL from 1 x
106 neutrophils per ml, measured for 30 minutes at 37 °C in hydrophobic tubes with 4U/ml
horseradish peroxidase and 5% (v/v) serum added. Experiments were performed in duplicate with
cells from three different donors. Bars indicates the standard errors of the means (SEM).
Figure 5. Neutrophil adhesion. Fluorescence from BCECF-labelled, adhered neutrophils
(rinsed samples) expressed as percent of the response on non-coated hydrophobic surfaces
(control). The fluorescence from non-rinsed (free and adherent cells) samples was 336% (±
23
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
23%). The autofluorescence from non-labelled cells was routinely subtracted. The
experiments were performed in triplicate with neutrophils from four donors. Bars indicate the
SEM. “IgG and NHS” was found to adhere significantly (p<0.05) more cells than all other
coatings. No other significant differences were found. NHS denotes the presence of 5%
normal human serum, and IgG indicates IgG-coated hydrophobic glass. Heat-inactivated
serum displayed similar results as NHS.
Figure 6. Neutrophils on hydrophobic glass with various protein coatings after 30 minutes
of incubation in 37 °C KRG. The neutrophils were fixed (4% paraformaldehyde) and F-actin
was stained (0.6 µg/ml bodipy-phallacidin, 100 µg/ml lysophosphatidylcholine), studied and
documented with a Sarastro 1000 confocal laser scanning microscope with a Nikon x 100
(1.4 N.A.) objective. Control indicates clean hydrophobic glass, NHS denotes presence of 5%
normal human serum, and IgG indicates IgG-coated hydrophobic glass. Heat-inactivated
serum gave rise to morphology similar to that with NHS.
24
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
Table 1: Effects of immobilized IgG on the radical production in diluted whole blood
No dilution
50% blood
25% blood
Hydrophobic glass (n=4)
995% (±302%) 1220% (±330%)
856% (±389%)
Hydrophilic glass (n=3)
669% (±148%) 352% (±44,1%)
168% (±45,4%)
25
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
Figure 1
26
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
27
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
Figure 2
28
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
Figure 3
29
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
Figure 4
30
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
Figure 5
31
“Complement activation on immunoglobulin G-coated hydrophobic surfaces...”
Figure 6
32